1. Field of the Invention
The invention relates to fusion proteins of Fc-binding domains and calcium-binding photoproteins, genes encoding the same, and use thereof. More specifically, the invention relates to fusion proteins of a ZZ domain having the ability to bind to the Fc region of immunoglobulin G (IgG) with the calcium-binding photoprotein apoaequorin, genes encoding the same and use thereof.
2. Related Art
Calcium-binding photoproteins are photoproteins in which an apoprotein and a peroxide of a light-emitting substrate exist in the state of a complex formed therefrom. Calcium-binding photoproteins have the property of emitting transient light on binding to a calcium ion.
Known calcium-binding photoproteins include aequorin, obelin, clytin, mitrocomin, mineopsin and bervoin. Of these, aequorin is a typical calcium-binding photoprotein, the higher-order structure and light-emitting mechanism of which have been reported in detail (see, for example, Inouye et al., Proc. Natl. Acad. Sci. USA, 82, 3154-3158 (1985); Head et al., Nature, 405, 372-376 (2000)). Since aequorin has a very high sensitivity to calcium ions, it is used to detect and assay trace amounts of calcium ions, and to measure changes in the intracellular concentration of calcium ions.
Aequorin exists in the state a complex formed from apoaequorin and a peroxide of the light-emitting substance coelenterazine. When aequorin bonds with a calcium ion, it momentarily emits light and forms coelenteramide, which is an oxide of coelenterazine, and carbon dioxide.
Protein A is a protein which originates in the cell wall of Staphylococcus aureus, and is known to bond specifically with the Fc region of IgG. It has been reported that a fusion protein of protein A and aequorin can be used in immunoassays. The minimum concentration of IgG that can be measured using a fusion protein of protein A and aequorin is 20 ng/mL per assay (see, for example, Biochem. Biophys. Res. Commun., 171, 169-174 (1990)).
The ZZ domain is a synthetic IgG-binding region which was developed based on the IgG-binding region of protein A (see, for example, Nilsson, B. et al., Protein Eng., 1, 107-113 (1987)). It has been reported that a fusion protein of the ZZ domain and the calcium-binding photoprotein obelin can be used in immunoassays. The minimum concentration of IgG that can be measured using a fusion protein of the ZZ domain and obelin is 10 ng/mL (see, for example, Biochem. Biophys. Res. Commun., 219, 475-479 (1996)).